LV11438B - Polymeric surfactant - encapsulated microbubbles and their use in ultrasound imaging - Google Patents

Abstract

PCT No. PCT/GB94/01923 Sec. 371 Date Mar. 14, 1997 Sec. 102(e) Date Mar. 14, 1997 PCT Filed Sep. 5, 1994 PCT Pub. No. WO95/06518 PCT Pub. Date Mar. 9, 1995The present invention relates to polymer-based gas-containing contrast agents in which microbubbles of gas are encapsulated by non-polymerizable wall-forming block or graft copolymer surfactants. The polymer surfactants are preferably biodegradable and include block and graft copolymers containing linkages of formula (I):-(O)m-CO-O-C(R1R2)-O-CO-(O)n-(I)where R1 and R2 each represent a hydrogen atom or a carbon-attached monovalent organic group, or R1 and R2 together form a carbon-attached divalent organic group and m and n are each zero or 1.

Description

LV 11438

POLYMERIC SURFACTANT-ENCAPSULATED MICROBUBBLES AND THEIR USE IN ULTRASOUND IMAGING

This invention relates to novel contrast aģents, more particularly to gas-containing contrast aģents of use in diagnostic imaging, and to novel polymers useful in thelr manufacture.

It 1s well known that ultrasonic imaging comprises a potentially valuable diagnostic tool, for example in studies of the vascular system, particularly in cardiography and of tissue microvasculature. A variety of contrast aģents has been proposed to enhance the acoustic images so obtained, including suspensions of solid pārticies, emulsified liguid droplets, gas bubbles and encapsulated gases or liquids. It is generally accepted that low density contrast aģents which are easily compressible are particularly efficient in terms of the acoustic backscatter they generate, and considerable interest has therefore been shovm in the preparation of gas-containing and gas-generating systems.

Gas-containing contrast media are also known to be effective in magnetic resonance (MR) imaging, e.g. as susceptibility contrast aģents which will act to reduce MR signal intensity. Oxygen-containing contrast media also represent potentially useful paramagnetic MR contrast aģents.

Furthermore, in the field of X-ray imaging it has been observed that gases such as carbon dioxide may be used as negative oral contrast aģents.

Initial studies involving free gas bubbles generated in vivo by intracardiac injection of physiologically acceptable substances have dēmonstratēd the potential efficiency of such bubbles as contrast aģents in echocardiography; such techniques are severely limited in practice, however, by the short lifetime of l the free bubbles. Interest has accordingly been ahown in methods of stabilising gas bubbles for echocardiography and other ultrasonic studies, for example using emulsifiers, olis, thickeners or sugars, or by entraining or encapsulatlng the gas or a precursor therefor in a variety of polymer systems, e.g. as gas containing polymer microparticles.

Thus, for example, W0 80/02365 discloses the use of gelatin encapsulated gas microbubbles for enhancing ultrasonic images. Such microbubbles do not, however, exhibit adeguate stability at the dimensions preferred for use in, echocardiography (1-10 μη) in view of the thinness of the encapsulating coating. US-A-4774958 discloses the use of microbubble dispersions stabilised by encapsulation in denatured protein, e.g. human serum albumin. Such systems permit the production of microbubble systems having a size of e.g. 2-5 pm but stili do not permit efficient visualisation of the left heart and myocardium. The use of such protein-derived aģents may also create problems with regard to potential allergenic reactions. EP-A-0327490 discloses, inter aJJLa, ultrasonic contrast aģents comprising a microparticulate synthetic biodegradable polymer containing a gas or volatile fluid (i.e. having a boiling point below 60°C) in free or bonded form. Representative synthetic biodegradable polymers include polyesters of hydroxy carbonic acids, polyalkyl cyanoacrylates, polyamino acids, polyamides, polyacrylated saccharides and polyorthoesters.

Similar biodegradable microparticulate polymere, based on polymerised aldehydes, are described in EP-A0441468, while systems based on microparticulate poly(amino acid)-poly(cyclic imide), derivatives are described in EP-A-0458079.

Ultrasonic contrast aģents consisting of microparticles consisting of amyloses or synthetic biodegradable polymers and a gas or volatile fluid are described in 89/06978. EP-A-0458745 discloses air or gas-filled microballoons in which the encapsulating material is a deformable and resilient interfacially deposited polymer which is preferably biodegradable, examples including polysaccharides, polyamino acids, polylactides, poly-glycolides, lactide/lactone copolymers, polypeptides, proteīns, polyorthoesters, polydioxanone, poly-P amino-ketonēs, polyphosphazenes, polyanhydrides and poly(alkyl cyanoacrylates). The microballoons are normally prepared by emulsion technigues leading to deposition of the 2 LV 11438 polymer around droplets of a volatile liquid which is 8ubsequently evaporated. Such techniques generally involve the use of surfactante, for example lecithins, fatty acids or estera thereof with polyoxyalkylene compounds such as polyoxyethylene glycol or polyoxypropylene glycol, in order to stabilise the emulsion.

It is generally acknowledged that polymer-based contrast aģents should desirably be biodegradable in order to facilitate their ultimate elimination from or absorption by the tēst subject. In many instances it has therefore been proposed to use polymers such as polyesters, polyanhydrides, polycarbonates, polyamides and polyurethanes which are biodegradable as a result of the eusceptibility of ester, amide or urethane groups therein to enzymic hydrolysis in vivo.

In our published International Patent Application No. W0 93/17718 there are described polymer-based contrast aģents which are designed to exhibit high and controllable Ievels of biodegradability in vivo by virtue of the presence in the polymer of methylene diester units of formula - (0)m-C0-0-C (RXR2) -0-C0- (0)n- 1 2 (where R and R each represent a hydrogen atom or a 1 2 carbon-attached monovalent organic group or R and R together form a carbon-attached divalent organic group and m and n are each zero or 1) . Such units are particularly rapidly degraded by common esterase enzymes but are relatively stable in the absence of enzymes. These polymers may be formulated into microparticle-and/or microballoon-containing contrast aģents by, for example, emulsion techniques such as those described in the above-mentioned EP-A-0458745. It will normai ly be necessary for a dispersing aģent, e.g. a surfactant, to be present during such Processing; provided that the surfactant is physiologically acceptable it may, if desired, be retained in the final product, for example to enhance the dispersibility and/or stability of the contrast aģent microparticles and/or microballoons in the intended carrier medium. W0 92/17212 describes polymer-based contrast aģents comprising microbubbles of gas or a gas precursor encapsulated by non-proteinaceous crosslinked or polymerised amphiphilic moieties, which may if desired contain methylene diester units of formula (I) as 3 described above. These contrast aģents may, for example, be prepared by-emulsifying a polymerisable amphiphile, e.g. so as to yield an oil-in-water emulsion in which a volatile vrater-immiscible organic solvent is encapsula-ted by the amphiphilic moieties, e.g. in the form o£ miceiIes, and thereafter' crossinking or polymerising the amphiphilic moieties and, if desired, removing the volatile organic solvent, e.g. by evaporation. It will be appreciated that the surface active properties of the polymerisable amphiphiles may render unnecessary the use of extraneous surfactants in the emulsification proce-dures of such processes. The polymerisable amphiphiles may, for example, themselves contain polymer groupings, e.g. hydrophilic polymer groups such as polvoxyethylene chains, as well as polymerisable groupings, e.ģ. (meth)acrylic or other poymerisable alkenyl or alkynyl groups, and hydrophobic groups such as long chain alkyl groups.

The present invention is based on the finding that polymer-based gas-containing contrast aģents in which microbubbles of gas are encapsulated by non-polymerisable wall-forming block or polymer surfactants possess a number of advantageous properties. Thus such contrast aģents may exhibit storage stability and good stability and contrast effect in vivo following ādministration, often for several passages of circu-lation in the case of ādminis tration by intravenous injection; they can be designed to undergo rapid biode-gradation thereafter. Moreover, it is possible by selection of the nature and size of the various reģions or domains of the block or graft copolymer to influence properties of the contrast aģents such as their stability, dispersibility, biological properties etc.

The fact that contrast aģents according to the invention may readily and easily be prepared directly from block or graft copolymer surfactants e.g. as described hereinafter, is also advantageous. Thus the use of prepolymerised wall-forming copolymer surfactant starting materiāls avoids the need for crosslinking or polymerisation reactions e.g. as are described in WO 92/17212, with the attendant need subseeņienty to remove by-products and/or residues from materiāls such as initiators from the products in order to render them physiologically acceptable; contrast aģents obtained directly from polymer starting materiāls may also exhibit greater structural integrity than contrast aģents obtained by crosslinking or polymerisation reactions. Furthermore, as a result of the surface 4 LV 11438 active properties of the block or graft copolymer surfactant starting materiāls, it is possible to prepare contrast aģents according to the invention by emulsification techniques without using extraneous surfactants/emulsifiers, although as will be described hereinater use of such emulsifiers is not precluded where desired in specific embodiments of the invention.

According to one aspect of the present invention there is provided a contrast aģent comprising gas microbubbles encapsulated by a non-polymerisable wall-forming block or graft copolymer surfactant.

The term "non-polymerisable" as used herein in respect of the copolymer surfactant indicates that this material will not normally undergo further polymeri-sation e.g. during preparation or use of contrast aģents according to the invention. It will be appreciated that the copolymer surfactant may, however, be capable of further polymerisation reactions under more extreme conditions than will be encountered in such circumstances.

The term "wall-forming" as used herein in respect of the copolymer surfactant indicates that this material is inherently capable of interacting to form an encapsula ting structure having a desired degree of integrity without reguiring Chemical reactions such as cros-slinking or further polymerisation to stabilise the structure. Such structures may, for example, take the form of solid micropar ticies, e.g. comprising one or more encapsulated gas microbubbles, or membranes or films encapsulating gas microbubbles diepersed in a liguid carrier.

The surfactant properties of copolymers used in contrast aģents according to the invention will normally arise from the presence in the copolymer of separate reģions or domains having different lyophilicity. Nost commonly one or more such reģions or domains will be hydrophilic and one or more other reģions or domains will hydrophobic, such that the copolymer exhibits amphiphilic properties. It may, however, also be possible to use copolymers containing separate reģions or domains exhibiting, for example, differing degrees of hydrophi1ic i ty.

Block copolymer surfactants which may be used in contrast aģents according to the invention include block copolymers having two or more blocks of differing lyophilicity, for example in linear di-block, tri-block or multi-block arrays, e.g. of the type A-B, A-B-A, B-A-A or A-B-A-B-A-B where A and B are polymer blocks of 5 differing lyoph±licity, e.g. hydrophilic and hydrophobic blocks respectively. Branched structurēs, e.g. of the type

B A-<

B and macrocyclic structurēs, e.g. of the type

A B may also be employed.

The size of one or other type of block may if desired be chosen to be relatively small in order to obtain a desired hydrophilic/lipophilic balance. Thus, for example, in the case of block copolymers containing hydrophilic and hydrophobic blocks it may be advantageous to select small-sizēd hydrophobic blocks in order to render the copolymer water-soluble.

In general where small-sized blocks are present these may include both oligomeric groups and quasi-polymeric groups, including monomeric groups, which may for βχβπφΐβ exhibit polymer characteristics (e.g. as a result of the presence of long chain units) while not strictly possessing a definable repeating unit. Copolymers containing such oligomeric or quasi-polymeric blocks are sometimes described in the art as wextended polymers". One category of such extended polymers useful in contrast aģents according to the invention comprises hydrophilic polymer blocks linked by oligomeric or quasi-polymeric hydrophobic reģions or domains.

Graft copolymer surfactants which may be used in contrast aģents according to the invention will normally comprise a first polymer having branches of a second polymer of different lyophilicity along its length; if desired either the first or second polymer may be a block copolymer, in which case the surfactant may be termed a block-graft copolymer. One useful type of graft copolymer surfactant comprises a hydrophobic polymer backbone having branches of a hydrophilic polymer along its length.

Copolymer surfactants in contrast aģents according to the invention may, for example, contain hydrophilic reģions or domains derived from polymers such as polysaccharides, polyalcohols (e.g. polyvinyl alcohol), polyvinylpyrrolidone, polyethylene glycol and polyaminoacids. Polymers such as polyorthoesters, 6 LV 11438 polyacetals, polyanhydrides, polyglycolic acids, poly-(meth)acrylic acids and derivatives such as estera thereof, substituted as necessary by hydrophilic groups, may also be useful. Contrast aģents comprising copolymer surfactants in which the hydrophilic reģions or domains consist essentially of polyethylene glycol units may be particularly advantageous.

The presence of charged groups within the hydro-philic reģions or domains may be advantageous since their high water-solubility may permit use of relatively small hydrophilic reģions or domains; interactions between such charged species may also enhance the stability of dispersions of the copolymer surfactant by inhibiting aggregation.

Hydrophobic reģions or domains in copolymer surfactants used in contrast aģents according to the invention may, for example, be derived from oil-soluble condensation, ionic and free-radical generated polymers, for example poly(meth)acrylate esters, polyorthoesters, vinylic and styrenic polymers, polyacetals, poly-anhydrides, polyglycolic acids and ethers and esters thereof, and polylactic acid/polyglycolic acid copoly-mers; such polymers may, for example, incorporate or be substituted with hydrophobic groups such as alkyl, aralkyl or aryl groups to increase their hydrophobicity. The hydrophobic reģions or domains may advantageously comprise a polyester Chain (which may be an oligomeric or quasi-polymeric moiety) containin one or more long chain aliphatic groups (e.g. C10_20 polymethylene groups).

The size of the hydrophobic blocks may particularly influence the wall-forming properties of copolymer surfactants which have low water-solubility, e.g. where these properties are (at least in part) the result of hydrophobic interactions. The physical State of the hydrophobic block, e.g. whether it tends to form crystal or amorphous structurēs and the degree of hardness or softness thereof, may also be of importance.

The different reģions or domains of the copolymer surfactant may be joined directly or through a linker, e.g. comprising a polyvalent atom or inorganic group or a multifunctional organic group such as a monomer unit occurring in one of the reģions or domains.

As has previously been noted, polymer-based contrast aģents should desirably be biodegradable in order to facilitate their ultimate elimination from or absorption by the tēst subject. The contrast aģents of the invention are therefore preferably biodegradable, i.e. comprise copolymer surfactants which incorporate 7 groups or bonds which are labila An vivo. The copolymer surfactants may therefore, for example, advantageously contain acid-labila bonds, e.g. as in polyorthoesters, polyacetals, polyanhydries, polyglycolic acids and ethers, esters and polylactic acid copolymers thereof. Other potentially useful copolymer components exhibiting biodagradability include polysaccharides, polyamino-acids, polylactides, lactide/lactone copolymers, poly-peptides, proteins, polydioxanones, poly-P-aminoketones, polyphosphazenes, and poly(alkyl cyanoacrylates). A particularly useful class of copolymer surfactants in contrast aģents according to the invention contain enzymically biodegradable methylene diester groups, e.g. of formula (I) as defined above. Examples of such grous are described in the aforementioned published international Patent Applications No. W0 92/04392 and W0 93/17718, the contents of which are incorporated herein by reference. 1 2

In such units of formula (I) R and R (when other than hydrogen) may, for example, each represent a carbon-attached hydrocarbyl or heterocyclic group, for example having 1-20 carbon atoms, e.g. an aliphatic group such as an alkyl or alkenyl group (preferably having up to 10 carbon atoms), a cycloalkyl group (pre- ferably having up to 10 carbon atoms) , an araliphatic group such as an aralkyl group (preferably having up to 20 carbon atoms), an aryl group (preferably having up to 20 carbon atoms) or a heterocyclic group having up to 20 carbon atoms and one or more heteroatoms selected from 0, S and N. Such a hydrocarbyl or heterocyclic grouping may carry one or more functional groups such as halogen 3 4 3 4 5 atoms or groups of the formula -NR R , -CON R R , -OR , - SR5 and -C00R6, where R3 and R4 are each hydrogen atoms 1 acyl groups or hydrocarbyl groups as defined for R and 2 5 R ; R is a hydrogen atom, an acyl group or a group as 12 6 defined for R or R ; and R is a hydrogen atom or a 12 12 group as defined for R or R . Where R and R represent a divalent grouping this may, for example, be an alkylidene, alkenylidene, alkylene or alkenylene group (preferably having up to 10 carbon atoms), which may carry one or more functional groups as defined above.

One preferred class of units of formula (I) 1 2 comprises those in which R and R are each selected from hydrogen atoms and methyl groups, e.g. in which R1 represents a hydrogen atom and R1 represents a methyl group. 8 LV 11438

If desired the properties of the wall-£orming polymer may be modified by a softener or elasticiser as described in EP-A-0458745.

Any biocompatible gas may be employed in the contraet aģents o£ the invention, for example air, nitrogen, oxygen, hydrogen, nitrous oxide, carbon dioxide, helium, argon, sulphur hexafluorlide and low molecular weight optionally fluorinated hydrocarbons such as methane, acetylene or carbon tetrafluoride. Use of perfluoroalkanes such as perfluorobutane or perfluoropentane may be advantageous. The gas, may be free within the encapsulating structure formed by the copolymer surfactant or may be entrained in a containing structure within the encapsulating structure. It will be appreciated that the term "gas" as used herein includes any substance which is in gaseous form at the normai human body temperature of 37°C.

The contrast aģents of the invention may if desired incorporate one or more additional emulsifiers, for example selected from fatty acids (e.g. straight chain saturated or unsaturated fatty acids, for example containing 10-20 carbon atoms) and carbohydrate and tri-glyceride esters thereof, phospholipids (e.g. lecithin), proteīns (e.g. albumins such as human serum albumin), polyethylene glycols and block copolymers (e.g. poly-(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) block copolymers such as Pluronics), including extended polymers. Block copolymer emulsifiers may, for example, have a similar cross composition to a block copolymer used as the wall-£orming polymer surfactant, but may exhibit a different hydropholic/lipophilic balance as a result of, for example, different ratios between the contents of hydrophilic and hydrophobic blocks.

Microparticulate contrast aģents of the invention may if desired be coated, for example with one or more coating materiāls such as polyethylene glycols, proteins or polysaccharides, e.g. to modify their aggregation tendencies and/or biological properties.

Contrast aģents according to the invention may be used in a variety of diagnostic imaging technigues, including ultrasound, MR and X-ray imaging. Their use in diagnostic ultrasonic imaging and in MR imaging, e.g. as susceptibility contrast aģents, constitute preferred features of the invention.

For ultrasonic applications such as echocardio-graphy, in order to permit free passage through the pulmonary system and to achieve resonance with the preferred imaging frequency of about 0.1-15 MHz, it may 9 be convenient to employ microbubbles having an average size of 0.1-10 μη, e.g. 1-7 μια. Substantially larger bubbles, e.g. with average sizes of up to 500 μια, may however be useful in other applications, for example gastrolntestinal imaging or Investlgations of the uterus or Fallopian tubes.

Mlcroparticulate contrast aģents according to the invention may, for example, be stored and transported in dry fona, in which condition they will normai ly be stable indefinitely, being mixed with an appropriate liguid carrier (e.g. sterile water for injection, physiological saline or phosphate buffer) prior to administration. In this way the concentration of the injected or otherwise ādministered contrast aģent may be varied at will, depending on the precise nature of the intended application. Contrast aģents of the invention may also be stored in suspension in such carriers, especially when the porosity of the encapsulating polymer membrane is comparatively low, and/or the encapsulated gas has low solubility in the carrier liquid.

The contrast aģents of the invention may be prepared by any convenient process. Such processes, which in general will involve interacting a gas with a non-polymerisable wall-forming block or graft surfactant so as to producē the desired contrast aģent, constitute a further feature of the invention.

Representative microencapsulation technigues for the preparation of materiāls encapsulated by a wall or membrane of polymer material are described in literature such as "Microencapsulation and Related Drug Processes" by P.D. Deasy, Marcel Dekker Inc., New York (1984).

Contrast aģents according to the invention may conveniently be prepared by emulsion techniques, e.g. such as are known in the polymer art. Such processes may typically involve (i) generating an emulsion comprising hydrophilic and hydrophobic phased wherein the copolymer surfactant is preferentially solubilised in the dispersed phase thereof or is distributed about the interfaces between said phases and (ii) obtaining the desired contrast aģent from the emulsion. Single or multiple emulsions may be generated; in the latter case the copolymer surfactant will desirably be preferentially solubilised in or distributed about the interfaces of the finest (i.e. innermost) of the dispersed phases. Representative multiple emulsion technigues are described in W0 93/11718. 10 LV 11438

The hydrophilic/lipophilic balance of copolymer surfactant starting materiāls may be selected to give copolymers appropriate to a particular form of emulsion Processing. Thus, for example, the Processing of oil-soluble copolymer surfactants using oil-in-water emul-sions may be advantageous. It may also be possible to use an oil-in-water emulsion to process a copolymer surfactant having a degree of water-solubility where the water-soluble blocks are such as to create strong attractive interactions which slow the kinetics of dissolution sufficiently to permit mitroparticle formation in the presence of water; a similar approach may be taken using a water-in-oil emulsion and a copolymer surfactant having a degree of oil-solubility.

Where it is desired to use copolymer surfactants which become distributed at the phase interfaces, e.g. in the form of a film or separate phase, it may be useful to employ copolymer surfactants capable of aggregation into liquid crystalline structurēs, e.g. as lamellar phases, hexagonal or reversed hexagonal phases, cubic phases, or other liguid or solid phases of copolymer surfactants in admixture with other components.

The hydrophobic phase of emulsions generated in accordance with this aspect of the process of the invention may, for example, comprise a water-immiscible organic solvent such as an aliphatic, cycloaliphatic or araliphatic hydrocarbon, e.g. containing up to 10 carbon atoms, for example n-octane, cyclooctane, a dimethyl-cyclohexane, ethylcyclohexane, a methylheptane, an ethylhexane, toluene, xylene or a terpene, terpenoid or isoprenoid such as camphene or limonene; a haloalkane, such as methylene chloride, chloroform, carbon tetrachloride, methyl bromide or a Freon; an ester such as ethyl or propyl acetate, butyl formate or propyl or isopropyl butyrate or isobutyrate, or an appropriate ether or other lipophilic solvent.

The emulsion may, for example, be prepared using conventional technigues such as agitation, sonication, stirring (preferably high speed stirring) or other forms of mixing (e.g. high shear mixing), the copolymer surfactant advantageously being predissolved in what is to be the dispersed phase. It will be appreciated that factors such as stirring speed will influence the size of the encapsulated microbubbles ultimately produced; thus, for example, faster stirring tends to yield smaller microbubbles. 11

One useful embodiment of this process according to the invention comprises generating a single or multiple emulsion vrtierein the copolymer surfactant is preferentially distributed about the (where appropriate innermost) interfaces between the phases, and removing either the dispersed phase or both phases, e.g. by evaporation, spray drying or, more preferably, lyoph.ilisation, to generate a desired microparticulate contrast aģent. The emulsion, which may for example be an oil-in-water, water-in-oil or water-in-oil-in-water emulsion, may advantageously be lyophilised or otherwise treated under an atmosphere of the gas which is to be incorporated in the contrast aģent, if desired at reduced pressure. Emulsifiers such as £atty acids and esters, phospholipids, proteins, polyethylene glycols and block copolymers (e.g. as hereinbefore described) may if desired be used in the emulsification process.

Where only the dispersed phase is removed in such a process the microparticles may be recovered from the dispersion phase by, for example, flotation or filtration.

In an alternative process a solution of the copolymer surfactant in an appropriate aprotic polar organic solvent (eg. a sulphoxide such as dimēthyl sulphoxide, a cyclic ether such as tetrahydrofuran or an N,N~disubstituted amide such as dimethylformamide) is mixed with an agueous phase (e.g. using a high speed stirrer) so as to precipitate polymer material which may be collected and lyophilised to yield microparticulate contrast aģents in accordance with the invention. The agueous phase may advantageously contain a polymer material such as polyvinyl alcohol ot poloxamer (e.g. a Pluronic). Such techniques are described in the abovementioned EP-A-0458079. A further process comprises injecting a solution of the copolymer surfactant in an appropriate organic solvent into liguid nitrogen; the solution may, if desired, also contain an additive such as hydroxypropylcellulose. Alternatively the copolymer surfactant may be dissolved in an appropriate solvent or dispersed in e.g. an oil-in-water, water-in-oil or multiple emulsion, and the solution or emulsion spray dried, e.g. as described in EP-A-0514790.

Coacervation techniques, e.g. as are known in the art, may also be employed in preparing contrast aģents according to the invention.

Contrast aģents of the invention may also be prepared by selecting copolymer surfactants having good 12 LV 11438 film-forming properties as a result of their hydro-philic/lipophilic balances and molecular sizes and which therefore have the ability to self-organise into micellar or liposome-like structures. Such copolymer surfactante may be used to prepare Solutions or dispersions of amphiphiles in laminar or lamellar fonn which may be used to generate gas-containing vesicles as described in W0 91/15244. Mixtures of copolymer surfactants with phospholipids (as well as synthetic biodegradable phospholipid derivatives of the copolymer surfactants, e.g. prepared as described by Laschewsky fit al in jL. Am. Chem. Soc. 109 (1987), p.788), optionally incorporating solubilising or viscosity-increasing aģents such as glycerol, propylene glycol, glucose or lactose, may also be used for the preparation of echogenic vesicles, e.g. as described in EP-A-0554213.

In a stili further variant of the process of the invention a gas-in-liguid emulsion is prepared in which the dispersed phase comprises the gas to be encapsulated and in which the copolymer surfactant is preferentially soluble in the liquid continuous phase, thereby generating a contrast aģent comprising a dispersion of gas microbubbles stabilised by encapsulating copolymer surfactant. In a preferred embodiment of this. process the gaseous dispersed phase is a hydrophobic perfluoro-alkane such as perfluorobutane or perfluoropentane and the dispersion phase is an agueous solution of a water-soluble copolymer surfactant, e.g. an extended polymer.

Copolymer surfactants useful in the preparation of contrast aģents according to the invention may, for example, be prepared by methods known in the art, e.g. as described in Polymer Surfactants by Ir ja Piinna (Surfactant Science Series Vol. 42 - Marcel Dekker, New York, 1992), the contents of which are incorporated herein by reference. Thus, for example, block copolymers may be prepared by technigues such as the active.end group approach, anionic polymerisation, cationic polymerisation or condensation of prepolymers. Graft copolymers may, for example, be prepared by copoly-merising a macromonomer comprising a polymer terminating at one end in a polymerisable group with a second monomer or by "grafting onto" or "grafting from" methods. Suitable block and block-graft copolymers are described in "Block Copolymers" by D.C. Allport & W.H. Janes, (Applied Sciences Publishers Ltd., London 1973).

The hydrophilic/lipophilic balance of the copolymer surfactants will be determined by factors such as the nature of the polymers constituting the different blocks 13 and the relative ratio between the total volumes of e.g. hydrophilic and hydrophobic blocks. This balance may therefore be selected to give polymers appropriate to a particular fona of emulsion Processing, e.g. as described above.

Block and graft copolymer surfactante containing biodegradable linkages of formula (I) as hereinbefore defined are themselves novel and constitute a further feature of the present invention, as do emulsions comprising such copolymer surfactants, e.g. emulsions comprising phases of-differing lyophilicity wherein the copolymer surfactant is preferentially solubilised in the dispersed phase thereof or is distributed about the interfaces betvreen said phases. Most commonly the phases will be hydrophilic and hydrophobic respectively, although the copolymer surfactants may also be used to stabilise oil-in-oil emulsions of immiscible oils.

The novel copolymer surfactants according to the invention may, for example, contain representative or 1 2 preferred atoms/groups R and R and hydrophilic and hydrophobic reģions or domains as hereinbefore described. The biodegradable linkages may for example comprise units of formula - (CH2)a- (Oi.-CO-O-CiRV) -0-C0- (0)n- (CH2)b- (II) 1 2 (where R , R , m and n are as hereinbefote defined and a and b are each integers in the range 1-30, preferably 10-18).

Preferred copolymer surfactants in accordance with this embodiment of the invention are block copolymers, including extended polymers. In addition to their usefulness as starting materiāls for the preparation of contrast aģents according to the invention these novel block copolymer surfactants containing biodegradable linkages of formula (I) may have a wide range of further Utilities, e. g. by analogy with knovm block copolymer surfactants (see for example Encyclopedia of Polymer Science Vol. 2 pp. 412-434- John Wiley and Sons, New York (1985).

Block copolymer surfactants in accordance with the invention may therefore find use in applications such as foods, e.g. in water-in-oil emulsions such as mayonnaise and margarine and oil-in-water emulsions such as synthetic milk and ice cream; paints, coatings and impregnations, e.g. as dispersing aģents for pigments or as biodegradable coatings to impart properties such as 14 LV 11438 water-resistan.ce or a glossy finish to materiāls such as paper or cardboard; cosmetics, e.g. moisturising cream and make-up; detergents, e.g. for general washing and cleaning as in laundry applications, or for dispersal o£ oil spills; phase transfer catalysts; drug formulations, e.g. as dispersants, solubilisers, gelling aģents and emulsifying aģents for drugs (including water-insoluble drugs). drug delivery systems, e.g. as carriers (including micellar carriers) to promote site-specific delivery and/or delayed release of drugs, where appropriate with concommitant reduction in toxic side effects; surgical materiāls such as implants, wound dressings, adhesives and the like, e.g. controlled release implants, implants for fracture fixation, tendon and ligament replacements, biodegradable dressings, dressings with controlled release, sutures, controlled release crearns and ointments, adhesives and bone cements; particle coatings, e.g. to provide targetting of an active therapeutic aģent to a desired bite such as the lymphatic system; coating of medical devices, e.g. to promote protein resistance; textiles, e.g. as anti-static aģents; thermoplastic elastomers (in contrast to conventional non-melt processable crosslinked elastomers} which may be used to manufacture slowly biodegradable replacement body parts such as blood vessels by thermal processing; biodegradable clear packaging films, wherein the domain sizes for the polymer blocks are less than the wavelength of visible light; polymer property modifiers, e.g. as dispersed pārticies in another polymer matrix, for example to modify the fracture properties thereof; self-lubricating materiāls, e.g. wherein biodegradation generates low molecular weight compounds which provide a lubricating effect; compatibilisers for polymer blende, e.g. to facilitate dispersion of one polymer in another (the bio-degradability of the copolymer surfactant may be utilised to accelerate degradation of relatively stable polymer blend); separation membranes exhibiting selec-tive transport properties e. g. as wound coatings per-mitting transport of gas to the wound but providing a barrier to dirt and infection; anti-fouling coatings, e.g. such that continuous controlled biodegradation from the surface prevents adhesion of organisms, secondary toxic components optionally being incorporated to provide further protection through controlled release thereof; foam-forming materiāls, e.g. for dispersing blowing aģents or producing biodegradable foams useful for introducing celis in, for example, bone marrow, 15 pancreas, liver and cartilage transplantē; antifoams, e.g. for use In machine dishwashing and the sugar beet industry; and in the manufacture of hydrogels, e.g. for use in the controlled release of drugs or agrochemicals, in cosmetics and toiletries and as highly absorbent materiāls, e.g. for nappies or spill containment.

The £ollowing non-limitative Examples serve to illustrate the invention.

List of abbreviations AIBN: 2,2'-azobisisobutyronitrile DMF: N,N-dimethylformamide DBU: 1,8-diazabicyclo [5.4.0] undec-7-ene (1,5-5) 6PC: gas phase chromatography

MgS04: magnesium sulphate

Mp: melting point PE6: polyethylene glycol THF: tetrahydrofuran SEC: size exclusion chromatography

Mw: weight average molecular weight

Mn: number average molecular weight EXAMPLE 1_-_Preparation of_intermediates and prepolymers a) Methylehe bis(16-hydroxyhexadecanoate)

To a solution of 16-hydroxyhexadecanoic acid (15.0 g, 0.055 mol) in DMF (200 ml), DBU (8.65 g, 0.055 mol) vas added at room temperatūra. After 5 minūtes with stirring, diiodomethane (7.37 g, 0.028 mol) was added. The mixture was left with stirring at room temperature for 2 days. DMF was evaporated under reduced pressure and the residue dissolved by adding chloroform (100 ml) and water (50 ml) . After separating the phases the agueous layer was extracted with chloroform (3 x 100 ml) and the combined organic phases were dried (MgS04) . The solvent was removed under reduced pressure and the residue recrystallised from ethyl acetate to give 10.17 g (65%) of the title product as a white solid. Mp: 96.2 °C. Sl NMR (300 MHz, CDC13) δ 1.2 - 1.4 (m, 44H), 1.5-1.6 (m, 8H), 2.35 (t, 4H), 3^64 (t, 4H), 5.75 (s, 2H). 13CNMR (75MHz, CDC13) : δ 24.43, 25.55, 28.81, 29.42, 32.63, 33.80, 62.91, 78, 112.20. 16 LV 11438 b) Acid chloride terminated poly(methyl methacylate 1000

Acid terminated poly(methyl methacrylate) was synthesised by a matched chain transfer polymerisation. 2-Ethoxyethanol (160 g) was heated to 120 °C. To this a mixture of 2-ethoxyethanol (80 g), methyl methacrylate (160 g, 1.6 mol), thioglycollic acid (14.4 g, 0.156 mol) and 4,4'-azobis(4-cyanopentanoic acid) (14.4 g, 0.1 mol) was added over a period of 1.5 hours. The reaction was held at 120 °C for a further 0.5 hours before cooling to room temperatūre. The polymer was precipitated into a cold solution of sodium chloride (5% w/v) . The polymer was purified by repeated precipitation from hot methanol into cold distilled water (3 times). End group analysis gavē a number average molecular weight of 910 Daltons.

Acid terminated poly(methyl methacrylate) (105g, 0.115 mol) waa dissolved in dry toluene (300 ml). The o solution was cooled to 0 C and oxalyl chloride (15 g, 0.118 mol), was added slowly. The reaction, as permitted to warm to room temperatūra, whereafter the excess oxalyl chloride was removed under reduced pressure to yield the title product C) <x_- Methyacryloyl -ω- methoxy (PEG) 2000

Dry a-hydroxy-oo-methoxy PEG 2000 (6.40 g, 3.20 mmol) was dissolved in THF (160 ml) and the solution cooled to 10 °C. Pyridine (0.38 g, 4.78 mmol) was diluted with THF (4 ml) and added to the solution under a dry nitrogen atmosphere. Methacryloyl chloride (0.50 g, 4.78 mmol), was diluted in THF (12 ml) and added dropwise. The temperature was gradually raised to room temperatūra and, the mixture was left with stirring for 24 hours. The reaction mixture was filtered and the remaining acid chloride and solvent removed under reduced pressure. The residue was dissolved in THF and precipitated by addition of ether to give 4.50 g (68%) of the title product. d) Bimetallic μ - Oxoalkoxide Catalyst:

Zn{QAl [CLCH (CHaI2l2i2

Following the method of US-A-3432445, anhydrous zinc acetate (23.00 g, 125.4 mmol) was added to a solution of aluminium isopropoxide (51.20 g, 250 7 mmol) 17 in decahydronaphthalene (130 ml). The mlxture was heated to 190 °C with stirrlng under nitrogen and the reaction allowed to proceed for three hours during which about 20 ml dlstlllate was collected in the boiling range 73-88 °C. The decahydronaphthalene was then removed under redu-ced pressure at 160-180 °C. The product, a resinous orange solid, was dissolved in distilled n-heptane and the solution centrifuged to remove any remaining solīds. A typical preparation gavē low yield (23%) and analysis for AI and Zn content gavē a mole ratio of Al/Zn of 1.98. e) Ethylidene bis [16-(5-chlorocarbonylpenta-noyloxy)hexadecanoate

In a three-necked:round bottomed flask eguipped with a reflux condenser, a glass gas inlet tube and a pressure equalizing dropping funnel was placed freshly distilled adipoyl chloride (2.60 ml, 17.50 mmol) dissolved in absolute chloroform (15 ml) . The temperature was raised to ca. 50 C and under a gentle stream of nitrogen through the solution, a solution of ethylidene bis(16-hydroxyhexadecanoate) (1.0 g, 1.75 mmol) in absolute chloroform (30 ml) was added dropwise and left at this temperature, a further 3 hours after addition. The mixture was then cooled to room temperature and quickly transferred into a 50 ml round bottomed flask egnipped for distillation under reduced pressure. Chloroform was first distilled off at normai pressure, then oil-pump vacuum was established and 0 excess adipoyl chloride distilled off at ca. 75 C, 5 mbar pressure, leaving the residual title compound (1.56g) . f) 16-Hexadecanoyloxyhexadecanoic_acid 16-Hydroxyhexadecanoic acid (5.43g, 19.9 mmol) was dissolved in tetrahydrofuran (190 ml) and pyridine (2.36 g, 2 9.9 mmol) was added. Palmitoyl chloride (5.48g, 19.9 mmol) was dissolved in tetrahydrofuran (10 ml) and added dropwise at room temperature. After stirring at room temperature for 16 hours, the mixture was filtered and the filtrate evaporated under reduced pressure. The residue was dissolved in chloroform, washed with, water (3 x 50 ml) , and the organic phase was dried (MgS04) . After evaporating under reduced pressure, the residue 18 LV 11438 was purified on a silica column, eluted with chloroform with increasing methanol concentration (from 1% to 2% methanol in chloroform) to give 8.41 g (83%) of the title compound. ^ NMR (300 MHz, CDC13), δ 0.85 (t, 3H, CH3) , 1.20-1.35 (s, 46H, -CH2-), 1.55 - 1.70 (m 6H, -CH2-), 2.25 ( t, 2H, -CH2-C(0)-O) , 2.45 (t, 2H, -CH2- COOH) , 4.05 (t, 2H, -0-CH2) . 13C NMR (75 MHz, CDC13)s δ 14.01, 22.57, 24.10, 24.91, 25.82, 28.53, 28.75, 28.94, 29.08, 29.15, 29.25, 29.36, 29.54, 31.81, 34.29, 35.16, 64.27, 76.48, 76.90, 77.101, 77.32, 169.50, 173.91. g) 16-Hexadecahoyloxyhexadecanoyl chloride 16-Hexadecanoyloxyhexadecanoic acid (7.73 g, 15.13 mmol) prepared as in above was dissolved in tetrahydrofuran (140 ml) and oxalyl chloride (4.80 g, 37.83 mmol) was added dropwise. The mixture was stirred at room temperature for 3 days and then the solvent and unreacted oxalyl chloride were evaporated under reduced pressure to give 8.0 g (100%) of the title compound. h) 1-[16-(16-Hexadecanoylohexadecanoyloxy)-hexadecanoyloxylethyl 16 -hydroxyhexadecanoate

Ethylidene bis (16-hydroxyhexadecanoate) (4.38 g, 7.67 mmol) vras dissolved in THF (80 ml) and pyridine (0.61g, 7.71mmol) was added. 16-hexadecanoyloxy- hexadecanoyl chloride (4.18 g, 7.90 mmol) vras dissolved in THF (20 ml) andladded dropvrise. After 3 days at room temperature the mixture vras filtered and the filtrate vras left at 20 °C for 2 hours. The precipitated product vras filtered and purified by flash chromatography (silicagel, chloroform) to give 2.4 g (29%) of the title compound. XH NMR (300 MHz, CDC13) : δ 0.85 (t, 3H, CH3) , 1.2-1.4 (s, 90H, -CH2-), 1.45 1 (d, 3H, -0-CH(CH3)-0-) , 1.5-1.7 (m, 14H, -CH2-), 2.25 (Μ, 8H, -CH2-C(O)-0-) , 3.60 (t, 2H, -CH2-OH), 4.05 (t, 4H, -C(O)-O-CHj-) , 6.85 (q, 1H, -0-CH(CH3) -0-) . 13C NMR (75 MHz, CDC13) δ 13.7, 19.1, 22.2, 24.2, 24.6, 25.2, 25.5, 28.2, 28.5, 28.7, 28.8, 29.0, 29.2, 31.5, 32.,3, 33.7, 34.0, 62.5, 64.0, 88.0, 171.5, 173.5. 19 i) Preparātiem af Methoxy-endcapped PEGs

Preparation of a Typical Polymer (MeO-PEG 2000)

An initiator solution was prepared by careful addition of potassium mētai (0.400 g, 10.23 mmol) to methanol (1.300 g, 40.57 mmol) in an inert atmosphere.

This initiator solution (0.220 g, 1.32 mmol potassium methoxide) was injected into an ampoule containing ethylene oxide (10.000 g, 227.00 mmol). The sealed ampoule was allowed to stand at room temperatūre o ovemight. The temperature was then raised to 60 C and reaction alloved for 72 hours. After removal of unreacted monomer, the contents of the ampoule were dissolved in dichloromethane and the solution neutralised with dilute acpieous hydrochloric acid. The polymer solution was washed three times with distilled water, rotary evaporated and then vacuum; dried. Assignments for MeO-PEG polymers. 1H-NMR: δ 2.7 (OH) , 3.2 (OCH3), 3.5 (-CH2- main chain), 3.4 (-CH2OCH3) . 13NMRs δ 58.5 (-OCH3), 61.2 (-CH2OH) , 70.5 (-CH2- main Chain), 71.3 (-CH2OCH3), 72.2 (-CH2CH2OH) .

The GPC was recorded in THF and the molecular weight calibration was via PEG standards. GPC data for a typical sample: Mp: 2679, Mn: 2012, Mw: 2283.

Polydispersity: 1.135. j) General-procedure for methoxy PEG chloroformate PEG 2000 monomethyl ether (6.00 g, 3.00 mmol) was dissolved in toluene (50 ml) and dried by refluxing in a Dean Stark apparatus. Pyridine (0.24 g, 3.00 mmol) was added at room temperature. Triehloromethyl chloroformate ("diphosgene") (0.60 g, 3.00 mmol) was dissolved in toluene (10 ml) and added dropwise. The mixture was stirred at room temperature for 12 hours and filtered. The solvent was evaporated under reduced pressure to give the title compound in guantitative yield. 20 LV 11438 ΕΧΑΜΡΙιΕ 2 - Preparation of Black and Grāft-copolymers

a) PEG 5QQ0-block-Polyester of-Methylene bis(16-hydroxyhexadecanoate & adipovl chloride-biock PEG 5 CLP Q

Methylene bis(16-hydroxyhexadecanoate) (0.56 g, 1.0 mmol) and dry α-methoxy-ω-hydroxy PEG of number average molecular veight 5000 (0.5 g, 0.1 mmol) were dissolved in a mixture of xylehe/trichloroethylene (80:20 100 ml), and heated to 60 °C. Adipoyl chloride (0.192 g, 1.05 mmol) was added. The mixtute-was refluxed at reduced pressure and 60 °C for 24 hours. The polymer was recovered by fractional precipitation from xylene/ trichloroethylene (80:20) at 4 °C. The product was shovm by SEC to have a Mn of 3800 and a Mw of 8000 (polystyrene eguivalents). b) EEG_2000-block-PolyeBter_of methylene bis (16-

hydroxyhexadecanoate) & adipoyl chloride-block-PEG 2 0 Q Q_ (Mefchod1)

Methylene bis(16-hydroxyhexadecanoate) (0.56 g, 1.0 mmol) and dry <x-methoxy-(o-hydroxy PEG of number average molecular weight 2000 (0.0572 g, 0.0286 mmol) were dissolved in a mixture of xylene/trichloroethylene (80:20 - 100 ml), and heated to 60 °C. Adipoyl chloride (0.186, 1.014 mmol) was added. The mixture was refluxed at reduced pressure and 60 °C for 24 hours. The polymer was recovered by fractional precipitation from xylene/trichloroethylene (80:20) at 4 °C. The product was shovm by SEC to have a Mn of 3400 and a Mw of 12700 (polystyrene equivalents). c) PEG_2000-block-Polyester_of methylene bis(16-hydroxvhexadecanoate & adipoyl chloride-blQ.ok.-EEG. 2Q0_Q (Method. 21

Methylene bis(16-hydroxyhexadecanoate) (0.56 g, 1.0 mmol) was dissolved in a mixture of xylene/tri-chloroethylene (80:20 - 100 ml), and heated to 60 °C.

Adipoyl chloride (0.201, 1.1 mmol) was added. The mixture was refluxed at reduced pressure and 60 °C for 35 minūtes. Dry a-methoxy-m-hydroxy PEG of number average 21 molecular weight 2000 (0.4 g, 0.2 mmol) was added to the reaction mixture. The mixture was re£luxed at reduced 0 pressure and 60 C for a further 24 hours. The polymer was recovered by fractional precipitation from xylene/trichloroethylene (80:20) at 4 °C. The product was shovm by SEC to have a Mn of 5200 and a Mw of 17500 (polystyrene equivalents). d) Bi-block copplymex._o£ Poly(methyl me.thacrylata). 1000 and PEG 2000 a-Methoxy-(D-hydroxy PEG of number average molecular welght 2000 (40 g, 0.02 mol) was dissolved In toluene and dried with molecular sieve (4 A) . To this a solutlon of acid chloride terminated poly(methyl methacrylate) as produced in Example 1(b) (20g, 0.02 mol) in toluene was added. The mixture was refluxed for 24 hours. The polymer was isolated by precipitation with petroleum ether (40-60) . The polymer was purified by ion exchange (IRA-400, Fisons) and by dissolution in water, heating above the cloud point of the copolymer and decantation (three times). The product so obtained was finally dissolved in toluene and precipitated with petroleum ether (40-60) to yield the title product as a white powder. e) Dl-block copolymer of Poly(methyl methacrylate) 1000 and PEG 4000 a-Methoxy-G)-hydroxy PEG of number average molecular weight 4000 (80 g, 0.02 mol) was dissolved in toluene and dried with molecular- sieve (4 A) . To this a solution of acid chloride terminated poly(methyl methacrylate) as produced, in Example l(b) (20 g, 0.02 mol) in toluene was added. The mixture was refluxed for 24 hours. The polymer was isolated by precipitation with petroleum ether (40-60) . The polymer was purified by ion exchange (IRA-400, Fisons) and by dissolution in water, heating above the cloud point of the copolymer and decantation (three times). The product so obtained was finally dissolved in toluene and precipitated:with petroleum ether (40-60) to yield the title product as a white powder. 22 LV 11438 £) Poly(Methvl methacrvlate)-grāft-PEG 2000 a-Methyacryloyl-<B-methoxy PEG of number average molecular weight 2000 as synthesised in Example 1(c) (0.50 g, 0.25 mmol) and AIBN (2 mg, 0.012 mmol) were dissolved In THF (3.0 ml) and degassed by repeated free-zing, evacuation and thawing cycles (4 times). Methyl methacrylate (0.5 ml, 4.7 mmol) was dietilled directly lnto the ampoule and the ampoule sealed. Polymerisation was performed in an oil bath at 60 °C for 22.75 hours.

The polymer was recovered by precipitation with Petroleum ether (40-60) then purified by dissolving in a small amount of THF and adding to water (200 ml) . On heating above 60 °C the polymer came out of eolutionThe title product was dried under reduced pressure. g) Multi-block copolymer of PEG and poly (methylene-bis (16-hydroxvhexadecanoate) plus adipoyl chloride 11x3;41 1) Polyester formation

Methylene bis(16-hydroxyhexadecanoate) (1.392 g, 2.50 mmol) was dissolved in a xylenel/trichloroethylene mixture (80:20), and heated to 60 °C. Adipoyl chloride (0.610 g, 3.33 mmol) was added. The mixture was refluxed at reduced pressure and 60 °C for 4 hours. 2) Coupling of blocks to give multi block copolymer

Dry α, ω di-hydroxy PEG 1500 (1.25 g, 0.833 mmol) was added to the reaction mixture from above. Refluxing at reduced pressure was continued for 2 days. The polymer was recovered by drying at reduced pressure. The residue was dissolved in dichloromethane and precipitated from methanol. SEC showed the product to have a Mn of 5600 and a Mw of 9400 (polystyrene eguivalents) and that there was no evidence of the presence of PEG homopolymer. MNMR indicated formation of a block copolymer, vith a molar composition of 2.6:1 polyester to poly(ethylene oxide). h) Di-block copolymer of PEG 2000 and Poly(lactic

acid) 2.QQQ <x-Hydroxy-o>-methoxy PEG 2000 (10.0 g, 5.0 mmol) was dissolved in toluene (300 ml) and dried by refluxing in a Dean and Stark trap for 12 hours. Poly(lactic acid) (Resomer L-104, Molecular weight 2000) (1.0 g, 0.5 mmol) and p-toluenesulfonic acid mono hydrate (2 mg, 0.001 23 mmol) were added. After Dean and Stark re£luxing of the mixture for three days, the eolvent was removed under reduced pressure, and the residue, as washed with water and filtered. i) Block copolymer of methvlene-bis (16-hydroxyhexade-. canoate). adipoyl chloride and

a-hydroxy-<o-inethoxv PEG 2Q0Q

Methylene bis(16-hydroxyhexadedandate) (8.0 g, 14.37 mmol) was dissolved in xylene/trichlorethylene (4:1) (250 ml) and heated to 60 °C. Adipoyl chloride (£reshly distilled) (2.92 g 15.97 mmol) was added dropwise and the mixture refluxed at 60 °C under vacuum (100 mbar) for 5 hours. Dry α-hydroxy-ω-methoxy PEG 2000 (6.39 g, 3.19 mmol) dissolved in toluene (58 ml) was added together with trichloroethylene (14 ml) . The mixture was refluxed under vacuum (100 mbar) for another 14 hours. After cooling to room temperature and precipitating in a refrigerator, the mixture was filtered. The precipitate vas dissolved in chloroform and precipitated once from hexane and then twice from methanol. A portion of the crude product (4.25 g) was dissolved in chloroform and precipitated once more from methanol to give the title compound (3.78 g) : NMR:200

Mhz δ: 1.3 (s, CH2) # 1.5-1.7 (m, CH2) , 2.2-2.4 (m, CH2C0), 3.6 (s, 0CH2CH20), 4.0 - 4.1 (m, CH20), 5.7 (s, 0CH20). SEC: Mp=21,191; Mn=5,571; Mw=21,079 (polystyrene equivalents). j) Block_copolymer_of methylene_bis(16-hydroxyhexa- decanoate). adipoyl chloride and g-hydroxy-ro-methoxy PEG 2000

Methylene bis(16-hydroxyhexadecanoate) (7.50 g, 13.47 mmol) was dissolved in xylene/trichlorethylene (4:1) (235 ml) and heated to 70 °C. Adipoyl chloride (freshly distilled) (2.74 g, 14.97 mmol) was added dropwise and the mixture refluxed at 70 °C under vacuum (100 mbar) for 5 hours. Dry a-hydroxy-£0-methoxy PEG 2000 (5.99 g, 2.99 mmol) dissolved in toluene (53 ml) was added together with trichloroethylene (13 ml) . The mixture was refluxed under vacuum (100 mbar) for another 40 hours. After cooling to room temperature and preci pitating in a refrigerator, the mixture was filtered. The precipitate was dissolved in chloroform and precipitated once from hexane and then once from 24 LV 11438 methanol. Hal£ the crude product wae purified by flash chromatography (silica, eluant: chloroform with stepwise increasing methanol concentration from 0 to 5%) giving the title compound (1.50 g) 1H KMR 300 MHz δ: 1.23 (s (br) , CH2) 1.57-1.65 (m, CH2) 2.31-2.36 (m, CH2CO), 3.37 (s, CH30) 3.63 (s/ 0CH2CH20) 4.01-4.06 (M, CH20) , 5.73 (S, 0CH20) SECsMp=13,313; Mn=6,357; Mw=12,351, (poly- styrene eguivalents). k) Elock_COPOlymer__ ethylidene bis (16-hydroxy-_ hexadecanoate). adipovl chloride and g-hydroxy-co-methoxY PEG 2000

Ethylidene bis(16-hydroxyhexadecanoate) (2.00 g, 3.50 imnol) was dissolved in xylene/trichlorethylene (4:1) (70 ml) and heated to 70 eC. Adipoyl chloride (freshly distilled) (0.73 g, 3.99 mmol) was added dropwise and the mixture re£luxed at 70 °C under vacuum (100 mbar) for 6 hours. Dry a-hydroxy-o-methoxy PEG 2000 (1.72 g, 0.86 mmol) dissolved in toluene (15 ml) vas added together with trichloroethylene (3 ml) . The mixture vas refluxed under vacuum (100 mbar) for another 40 hours. After cooling to room temperatūra and precipitating in a refrigerator, the mixture vas filte-red. The precipitate vas dissolved in chloroform and purified by flash chromatography (silica, eluant: chloroform with 0.75% methanol) to give the title compound (0.42 g). NMR 300 MHz δ: 1.24. (s (br), CH2), 1.44 (d, CH3-CH), 1.59-1.64 (m, CH2), 2.26-2.31 (m, CH2CO), 3.37 (s, CH30), 3.64 (s, 0CH2CH20); 4.04 (t, CH20), 6.85 (q, CH). SEC: Mp=12,410; Mn=3,830; Mw = 8,715 (polystyrene eguivalents). 1-r) Precaration _of _ Di-block Copolvmers:

General Procedure

The di-block copolymers vēre prepared by sequential polymerization of ethylene oxide (EO) with caprolactone (CPL), of ethylene oxide with lactide (LD), and of ethylene oxide with caprolactone and lactide. The catalyzed, by the bimetallic μ-οχοβίλοχίάβ was prepared in Example 1(d).

The dry solvent, toluene or tetrahydrofuran, was distilled, into a fleuned glass ajnpoule. Ethylene oxide was dried over calcium hydride and condensed into the glass eunpoule under high vacuum. An appropriate quantity 25 of catalyst, to give an ethylene oxide block of the desired length, was added by injection into the ampoule under a nitrogen atmosphere. The ampoule was sealed, heated to 60 °C and the reaction allowed to proceed for 24 hours. Caprolactone, lactide, or caprolactone and lactide mixtures In tetrahydrofuran were next added by injection of an appropriate quantity into the ampoule under a nitrogen atmosphere. The ampoule was heated to 60 °C for 24 hours, and then the polymerization was terminated by addition of isopropyl amine. The polymer solution was diluted with tetrahydrofuran and washed with dilute aqueous citric acid to remove catalyst residues. The polymer solution was further washed with distilled water until neutral and the high molecular weight polymer precipitated with n-heptane. The precipitated polymer vas finally vacuum dried to yield a white polymer. The nature of the polymerizations performed and results are summarized in Table 1.

Table 1

Table 1

Ex. Monomers parts by weight EO CPL LD Diluent Cat/Mon mol % Conv % MW by "H-NMR 1 1.0 7.6 toluene 0.26 88 EOl.OOO/CPLS ,700 a 1.0 1.1 toluene 0.30 93 E02,500/CPL2,800 n 1.0 3.0 THF 0.32 74 E04,0OO/CPL12,000 o 1.0 1.7 toluene 0.33 68 E03,000/CPL5.900 P 2.0 7.4 1.0 THF 0.44 48 E02 > 400/(CPL8,600 -LD700 q 1.7 1.0 TBF 1.69 49 E06/400/LD900 r 1.4 1.0 THF 1.82 56 E03,500/CPL500 S) Polymerization catalyzed by aluminium isopropoxlde

Aluminium isopropoxide (1.19 g, 5.8 mmol) was dried under high vacuum in a glass ampoule for 4 hours at room temperatūra. Dry toluene was distilled into the ampoule under high vacuum and dissolution of the aluminium isopropoxide allowed. Ethylene oxide (7.42 g, 168.6 mmol) was dried over caļcium hydride and condensed into the glass ampoule under high vacuum. The ampoule was sealed, heated to 45 °C and reaction allowed to proceed for 39 hours. Caprolactone (8.63 g, 75.6 mmol) was next added by injection into the ampoule under a nitrogen atmosphere. The ampoule was heated to 45 °C for a further 24 hours. The highly viscous product was dissolved in dichloromethane, the polymerization terminated with dilute agueous acetic acid and the high molecular weight 26 LV 11438 polymer precipitated with n-heptane. Molecular weight was determined by ^H-NMR indicating ethylene oxide and caprolactone block lengths at 500 and 5,200, respectively. t-y) Preparation of Poly(Methyl Methacrylate) - craft- PEGs: General procedūra a-Methacryloyl-(D-methoxy PEG 2,000 as synthesised in Example 1(c) was added to AIBN in a glass reactor and then evacuated. Toluene (ca. 4Omi) was distilled under vacuum into the reactor followed by destabilised methyl methacrylate (MMA) monomer. The reactor was sealed and then heated to 50 °C for approxixnately 20 hours. The tītie polymer was recovered by precipltation into heptane. It was purlfied by dissolving in a minimum amount of THF, addlng this solution to water and then heatlng above the cloud point of the polymer, at which polnt the polymer came out of solution. This was repeated once. The prepared graft copolymers were characterised by ^-NMR (Table 2):

Table 2

Ex Methacryoyl-PEG (g) MMA monomer (ml) AIBN (mg) NMR Analysis PEG content (wt—%) t 4.0 4.0 6.6 34 u 2.0 4.0 6.6 24 V 4.0 2.0 3.5 50 w 3.0 4.5 8.1 27 X 4.5 3.0 5.7 41 y 0.5 10 16.5 1.3 z) Random chain-extended polymer of-PEG 1500. adipoyl chloride and ethylidene bis(16-hydroxyhexa-decanoate) (0.37:1. 85:1.75). multiblock

To a suspension of ethylidene bis(16-hydroxyhexa-decanoate) (1.0 g, 1.75 mmol) in dimethoxyethane (10 ml) at room temperature was added freshly distilled adipoyl chloride (270 μΐ, 1.85 mmol). The temperature of the mixture, was gradually raised to 60 °C, and a colourless solution obtained. After 5 hours at this temperature PEG 1500 (0.55 g, 0.37 mmol) vras added and heating continued for a further 17 hours before the mixture was cooled to room temperature, the solvent evaporated and the solid 27 residue stirred in petroleum ether (bp 40-60 °C) for 15 minūtes and filtered to give the title compound (1.30 g) as a white solid. aa) Extended polymer from PEG 1500 and ethylidene.-bis Γ16- (5-chlorocarbonvlpentanovloxv)-hexadecanoatel(A-B-A)

Ethylidene bis[16-(5-chlorocarbonylpentanoyloxy)-hexadecanoate] prepared as in Example l(e) (0.88 g, 1.02 mmol) was dissolved in toluene (15 ml) in a 100 ml 3-necked round bottomed flask eguipped with a glass gas inlet tube and a reflux condenser. PEG 1500 (3.06 g, 2.04 mmol) was added and the mixture heated at 60 °C for 22 hours, cooled to room tempera tūre and the solvent removed under reduced pressure to give the title compound (4.12 g), as a white wax. ab) Extended polymer_from ..PEG_150 0 and_ethylidene . bis Γ16-(5-hlorocarbonYlpentanoyloxy)hexadecanoate1 (multiblock)

The reaction was performed as in Example 2 (aa), but with ethylidene bis [16-(5-chlorocarbonylpentanoyloxy)-hexadecanoate (1.02 g, 1.18 mmol) in toluene (20 ml) and PEG 1500 (1.77 g, 1.18 mmol) to give the title compound (2.29 g) as a white wax. ac-af) Extendet polymer of PEG, adipic acid and ethylidene bis(16-hydroxyhexadecanoate) (random multiblock)) A solution of PEG 2000 (A) (4-14 g, 2.07 mmol) in 1,1,2-trichloroethylene (26 ml) was added via a syringe to a round bottomed flask containing ethylidene bis(16-hydroxyhexadecanoate) (B) (118 mg, 0.207 mmol), under nitrogen atmosphere. The'resulting mixture wad heated to 60 °C, and when a clear solution had been obtained, adipoyl chloride (C) (417 mg, 2.277 mmol) was added via a syringe. The pressure was reduced to 250 mbar and the solution was stirred at 60 °C over a period of 92 hours. Remaining hydrogen chloride, evolved in the reaction, and the solvent were removed on a rotatory evaporator at reduced pressure and 60 °C for 3 hours, and subsequently under vacuum (<0.1 mm Hg) at 60 °C for 24 hours. Finally, the polymer was precipitated from an acetone solution by adding petroleum ether, and cooling in an 28 LV 11438 ice bath for 2 hours. Filtration yielded 3.5 g of the polymer as a white waxy solid.

In total four different block copolymers differing in the molecular weight of the starting PEGs were prepared by this method; the conditions specific for each polymerisation are given in Table 3 below. 13C NMR-and XH NMR-spectra of the polymers was in agreement with the expected products.

Table 3

Entry MW for starting PEG Molar ratio AīBsC1 Solvent Reaction time (hours) ac 400 10:1:11 Diglyme -xylene 21 ad 600 10:1:11 Diglyime 24 ae 1500 10:1:11 DME 21 af 2000 10:1:11 Trichloro- ethylene 92 ) The letters refers to the reactants as specifed in the text above. ag) PEG 2300 methyl ether 16-hexadecanoyloxyhexadecanoate PEG 2300 methyl ether (10.000 g, 4.35 mmol) was dissolved in tetrahydrofuran (90 ml) and pyridine (0.413 g, 5.22 mmol) was added. 16-hexadecanoyloxyhexadecanoyl chloride (2.301 g, 4.35 mmol) was dissolved in tetrahydrofuran (10 ml) and added dropwise. After stirring tor 3 days at room temperatūre, the mixture was filtered and the solvent was evaporated under reduced pressure. The residue (12.08 g) was purified on a silica column, eluted with chloroform with increasing methanol concentration (from 1% to 3% methanol in chloroform) to give 5.20 g (43%) of the title Compound. ^ NMR (300 MHz, CDC13) δ 0.80-0.87 (M, CH3), 1.21 (s, (br) , CH2), 1.53- 1.62 (m, CH2) 2.20-2.35 (m, CH2C0) , 3.34 (s, CH30), 3.61 (s, 0CH2CH20) , 4.02 (t, C00CH2ZH20) 4.19 (t, C00£H2CH20) 13C NMR (75 MHz, CDC13) s 6 13.95, 22.49, 24.71, 24.83, 25.74, 28.45, 28.95, 29.07, 29.16, 29.28, 29.34, 29.40, 29.46, 31.72, 34.05, 34.21, 58.85, 63.15, 64.19, 69.01, 70.37, 71.73, 173.64, 173.82. 29 ah) PEG 5000 methvl ether 16-hexadecanovloxyhexadecanoate) PEG 5000 methyl ether (7.500 g, 1.50 mmol) was dissolved ln toluene (90 ml) and dried by re£luxing in a Dean Stark apparatus. Pyridine (0.143 g, 1.80 mmol) was added followed by addition (dropwise) of 16-hexadecanoyloxyhexadecanoyl chloride (1.191 g, 2.25 mmol) dissolved in toluene (10 ml) . The mixture was heated to re£lux and after stirring under reflux for 3 days the mixture was cooled to room temperature and precipitated into hexane. After filtering, the precipitate was washed with hexane and dried (MgS04) . After evaporation under reduced pressure, the residue was purified on a silica column, eluted with chloroform with increasing methanol concentration (from 1% to 3% methanol in chloroform) to give 5.93 g (72%) of the title compound 1H NMR (300 MHz, CDC13) : δ 0.82-0.86 (m, CH3), 1.22 (s/ (br), CH2), 1.53-1.62 (m, CH2) , 2.20-2.35 (m, CH2CO), 3.34 (s, CH30), 3.61 (s, 0CH2CH20) , 4.01 (t, C00CH2CH20), 4.18 (t, COO£H20) . 13C NMR (75 MHz, CDC13) : δ 13.66, 22.21, 24.43, 24.54, 25.46, 28.17, 28.61, 28.79, 28.87, 28.99, 29.06, 29.11, 29.17, 31.44, 33.73, 33.93, 58.57, 62.87, 63.90, 68.72, 69.62, 69.86, 70.09, 71.45, 76.85 173.35 173.53. ai) PEG 10000 methyl ether 16-hexadecanoyloxyhexa- decanoate PEG 10000 methyl ether (7.500 g, 0.75 mmol) was dissolved in toluene (140 ml) and pyridine (0.107 g, 1.35 mmol) was added. The solution was heated to 60 °C and 16-hexadecanoyloxyhexadecanoyl chloride (0.595 g, 1.12 mmol) dissolved in toluene (10 ml) was added dropwise. The mixture was heated to reflux and after stirring under reflux for 3 days the mixture was cooled to room temperature and precipitated into hdxane. After filtering, the precipitate was washed with hexane and dried. Flash Chromatography on a silica column, eluted with 5% methanol in chloroform, gavē 5.399 (68%) of the title compound. ^ NMR (300 MHz, CDC13) : δ 0.84 (t, CH3) 1.21 (s, (br), CH2) , 1.55-1.60 (m, CH2) , 2.20-2.35 (m, CH2CO), 3.34 (s, CH30), 3.61 (s, 0CH2CH20), 4.01 (t, COOCH2CH20) , 4.18 (t, C00CH2CH20) . 13C NMR (75 MHz, CDC13): δ 13.94, 22.48, 24.70, 24.82, 25.73, 28.94, 29.05, 29.14, 29.26, 29.33, 29.39, 29.45, 31.71, 34.00, 30 LV 11438 58.84, 63.14, 68.99, 69.36, 69.86, 69.97, 70.01, 70.36, 70.74, 70.82, 70.86, 71.72, 77.10, 173.62, 173.80. aj ) 16-Γω-Methoxy-PEG 2000-earbonyloxylhexadecanoic acid 1-Γ16-(16-hexadecanoyloxyhexadecanoyloxy)-hexadecanoyloxv)ethvl ester

Methoxy PEG 2000 chloroformate (1.90 g, 0.95 mmol) was dissolved in toluene (90 ml), and pyridine (0.099, 1.13 mmol) was added. 1[[16 -(16-hexadecanoyloxyhexade-canoyloxy)hexadecanoyloxy]ethyl 16-hydroxyhexadecanoate (1.00 g, 0.95 mmol) was dissolved in toluene (10 ml) and added dropwise. The mixture was heated to reflux and after stirring under reflux for 10 hours, the mixture was cooled to room temperature and filtered. The solvent was evaporated under reduced pressure. The residue was purified on a silica column using chloroform containing 2% methanol, to give 1.00 g (35%) of the title compound. *Η NMR (300 MHz, CDC13) : δ 0.85 (t, CH3) , 1.20-1.33 (m, CH2) , 1.45 (d,-0-CH(CH3) -O) , 1.5-1.7 (m, CH2) , 2.0 (H20) , 2.2-2.3 (m, -CH2-C(0)-0), 3.35 (s, CH3-0-), 3.5-3.7 (s, -0CH2CH20-), 4.03 (t, -C(O) -0-£H2-) 4.10 (t, -£H2-0-C (O) - 0-), 4.26 (M,-O-C(O)-0-CH2-CH2-0-) , 6.8 - 6.9 (q, -0-£H(CH3-0) . 13C NMR (75 MHz, CDC13) : 6 13.7, 19.2, 22.1, 24.2, 24.6, 25.2, 25.5, 28.2-29.2, 31.5, 33.91 34.0, 58.7, 64.0, 66.3, 67.9, 68.5, -70.0, 71.5, 87.9, 171.5, 173.7. ak) 16- [Q? - Me thoxy - PEG 5000-carbonyl·oxyl·hexadecanoic acid 1- Γ16- (16-hexadecanoyloxyhexadecanoyloxy) -hexadecanoyloxyļethyl ester

Methoxy PEG 5000 chloroformate (8.50 g, 1.70 mmol) was dissolved in toluene (90 ml) and pyridine (0.146 g, 1.85 mmol) was added. 1-[16-(16-Hexadecanoyloxyhexa-decanoyloxy)hexadecanoyloxy]ethyl 16-hydroxyhexadecanoa-te (1.79 g, 1.70 mml) was dissolved in toluene (10 ml) and added dropwise. The mixture was heated to reflux and after stirring under reflux for 3 days the mixture was cooled to room temperature and filtered. The solvent was evaporated under, reduced pressure and the residue was purified on a silica column, eluted with chloroform with increasing methanol concentration (from 3% to 5% methanol in chloroform) to give 3,90 g (38%) of the title compound. *H NMR (300 MHz, CDC13) : δ 0.85 (t, CH3) 1.20-1.33 (m, CH2) , 1.45 (d, -O-CH (CH3)-0) , 1.5-1.7 (m, CH2), 1.8 (H20), 2.2-2.3 (m, -CH2-C(O)-O), 3.35 (s, CH3- O-), 3.5-3.7 (s, -0CH2CH20-), 4.03.(t, -C(0)-0-CH2-), 4.10 (t, -CH2-0-C(0)-0-) , 4.26 (m, -O-C (0)-0-CH2-CH2-0-) , 6.8-6.9 (q, -0-CH(CH3)-O) . al) 16- fco-Methoxy-PEG 10000-carbonyloxylhexadecanoic acid 1-[16-(16-hexadecanoyloxyhexadecanoyloxy)-hexadecanoyloxylethyl eeter

Methoxy PEG 10000 chloroformate (7.50 g, 0.75 mmol) was dissolved in toluene (90 ml), and pyridine (0.063 g, 0.80 mmol) was 1-[16-(16-hexadecanoyloxyhexadecanoyl-oxy)hexadecanoyloxy]ethyl 16-hydroxyhexadecanoate (0.79 g, 0.75 mmol) was dissolved in toluene (10 ml) and added dropvrise. The mixture was heated to reflux and after stirring under reflux for 3 days the mixture was cooled to room temperatūre and filtered. The solvent was evaporated of under reduced pressure. The residue was purified on a silica column, eluted with chloroform with increasing methanol concentratioh(from 3% to 5% methanol in chloroform) to give 1.60 g (19%) of the title compound. ^ NMR (300 MHz, CDC13) : δ 0.85 3 (t, CH3) 1.20-1.33 (m, CH2) , 1.45 (d, -O-CH (£H3)-0) , 1.5-1.7 (m, CH2), 2.2-2.3 (m, -CH2-C(0)-0), 3.35 (s, CH3-0-) , 3.5-3.7 (s, -0CH2CH20-), 4.03 (t, -C(0)-0-CH2) , 4.10 (t, CH2-0- C(O)-O-), 4.26 (m, -0-C(0)-0- CH2-CH2-0-), 6.8-6.9 (q, -0-CH(£H3) -0) . EXAMPLE 3 - Preparation of polymer pārticies a-e) General procedure A solution of copolymer in toluene was prepared. This solution was added to water (25-30% toluene v/v) and mixed with a high speed mixer (20500 rpm for 40 to 60 seconds). The resultant emulsion was freeze dried resulting in a fine white powder.

Example Polymer from Example 2 Concentration of copolymer in Toluene (wt/wt) 3 a a 10 3 b b 10 3 c c 10 3 d d 10 3 e e 10 LV 11438 £) Pārticies from polymer of Example 2(f)

Polymer from Example 2(£) (70 mg) was dissolved in toluene (1 ml) and added to water (3 ml) . The mixture was hand shaken for 30 seconds, frozen with dry ice/ methanol and £reeze dried. g) Pārticies from polymer of Example 2 (q)

Polymer from Example 2. (g) (100 mg) was dissolved in toluene (1 ml) and added to water (3 ml), The mixture was hand shaken for 30 seconds, frozen with dry ice/ methanol and freeze dried. h-i) Pārticies from AB block copolymers of PEG. polycaprolactone and poly(lactic acid)

General procedure: λ solution of the block copolymer was prepared in toluene. Ca. 2 ml of solution was added to 10 ml of water with, in some cases, an extraneous emulsifier dissolved in the water. The mixture was mixed with a high speed mixer. The sample was immediately frozen and freeze dried to give a fine white powder.

Example Block copolymer from Example No/concentra-tion Extraneous emulsifier Mixer speed (rpm) Mixer time (seconds) 3 h 2p/13 wt % 2.5 wt% pluronic F68 8,000 15 3 i 2p/13 wt % 1.25 wt% pluronic F68 8,000 15 3 j 2p/13 wt % 0.63 wt% pluronic F68 8,000 15 3 k 2p/10 wt % none 8,000 15 3 1 2n/10 wt % none 8,000 15 33 m-p) Pārticies from Polv(Methyl Methacvlate)_- graft EEGfi

General Procedure Λ solution of the graft copolymer was prepared in toluene. Ca. 2 ml of solution was added to 10 ml of water with, in some cases, an extraneous emulsifier dissolved in the water. The mixture vas mixed wit a high speed mixer. The sample was immediately frozen and freeze dried to give a fine white powder.

Example Graft copolymer from Example No/concentra-tion Extraneous emulsifier Mixer speed (rpm) Mixer time (seconds) 3 m 2t/4 wt % and 2y/6 wt % none 20500 30 3 n 2w/5 wt % and 2y/5 wt % none 8,000 305 3 o 2w/6 wt % and 2y/4 wt % none 20500 30 3 P 2w/5 wt % and 2 y/5 wt % 1. Owt % Pluronic F68 8,000 30 g) Pārticies from block copolymer of methylene bis(16-hvdroxvhexadecanoate). adipoyl chloride and q-hvdroxY-m-methoxy PEG 2000

The polymer of Example 2 (i) (0.1 g) was dissolved in 1.9 g toluene and mixed for one minūte with 3 ml water using an Ystral mixer at 10,000 rpm to form a water-in-oil emulsion. The water-in-oil emulsion was then emulsified in water (3 ml) to form a water-in-oil-in-water emulsion, which was freeze dried to give air-filled pārticies. r) Pārticies from block copolvmer of methvlene bis (16-hydroxyhexadecanoate). adipovl chloride and g-hydroxy-(Q-methoxy PEG 2000

The procedure of Example 3 (q) was repeated except that the second water phase of the water-in-oil-in-water emulsion contained 2% gelatin. s) Preparation of microbubbles of extended polymer from Eacan^ple 2 (ab) filled with perfluoro-n-butane (by shafring) 34 LV 11438

Polymer from Example 2 (ab) (0.02 g) was dissolved in distilled water (1 ml) . The solution vas degassed, and the headspace of the vessel was filled with perfluoro-n-butane. The solution was shaken for 18 seconds on a Capmix . Perfluoro-n-butane-filled micro-bubbles of a size suitable for intravenous ādmini -stration were observed by microscopy. The microbubbles were stable for several days. t) Preparation of microbubbles of extended polymer from Example 2 (ab) filled with perfluoro-n-butane (by aonlcatingr)

Polymer from Example 2 (ab) (0.25 g) was dissolved in distilled water (5 ml). The solution was degassed and then treated with a sonicator for 1 minūte under a stream of perfluoro-n-butane. Perfluoro-n-butane-filled microbubbles of a size suitable for intravenous ādmini stration were observed in a microscope. The microbubbles were stable for several days. u) Preparation of microbubbles of extended polymers from Examples 2 (ab) and 2 (ai) filled with perfluoro-n-butane (by shaking)

Polymer from Example 2 (ab) (0.01 g) was dissolved in distilled water (0.5 ml) and added to 0.5 ml of an agueous solution (1%) of the polymer from Example 2 (ai) . The solution was degassed, and the headspace of the vessel vas filled with perfluoro-n-butane and shaken for 99 seconds on a Capmix . Perfluoro-n-butane-filled microbubbles of a size suitable for intravenous ādmini stration were observed in a microscope. The microbubbles were stable for several days. v) Preparation of microbubbles of-Hypermer B 246 filled with perfluoro-n-butane (by shaking) ®

Hypermer B246 (0.01 g) (ICI) was dissolved in 1 ml of a solution of 4.1% (weight) glycerol and 1.4% (weight) propylene glycol in water. The sample vas degassed, and the headspace of the vessel filled with pertluoro-n-butane and shaken for 99 seconds on a ®

Capmix . Stable perf luoro-n-butane-filled microbubbles of a size suitable for intravenous ādministration were observed in a microcope. 35 EXAMPLE Acoustic characterisations (in-vitro)

General procedure

TM

The samples were re-dispersed in HilliQ water by shaklng on a laboratory shaker for an appropriate time. The dispersions were, then observed by light microscopy to determlne the particle size.

Example Pārticies from Example 3 Particle size (μιη) 4 a a 3-35 4 b b 5-70 4 c c 3-15 4 e e 1-5 4 £ £ 50-70 4 g g 1-5 4 h h 2-12 4 I I · 4-10 4 j • D 3-12 4 k k 4-12 4 1 1 1-12 4 m m 2-25 4 n n 4-20 4 o o 2-40 4 P P 2-44

The pārticies were characterlsed by measuring the ultrasonic tramsmission through agueous dispersions o£ the pārticies using a 3.5 MHz broad band transducer in a pulse-reflection technigue. The aqueous solvent was used as a reference. 36 LV 11438

Example Pārticies from Example 3 Result 4 a a Contrast 4 b b Contrast 4 c c Contrast 4 e e Contrast 4 £ f Contrast 4 g g Contrast 4 h h Contrast 4 I I Contrast 4 j j . Contrast 4 k k Contrast 4 1 1 Contrast 4 m m Contrast 4 n n Contrast 4 o o Contrast 4 P P Contrast 37 LV 11438

Claims 1. A contrast aģent comprising gas microbubbles encapsulated by a non-polymerisable wall-forming block or graft copolymer surfactant. 2. A contrast aģent as claimed in claim 1 wherein the copolymer surfactant contains one or more hydrophilic reģions or domains and one or more hydrophobic reģions or domains. 3. A contrast aģent as claimed in claim 2 wherein the copolymer surfactant contains one or more hydrophilic reģions or domains selected from polysatcharides, polyalcohols, polyvinylpyrrolidones, polyethylene glycols and polyaminoacids. 4. A contrast aģent as claimed in claim 3 wherein the copolymer surfactant contains one or more hydrophilic reģions or domains consisting essentially of polyethylene glycol units. 5. A contrast aģent as claimed in any of claims 2 to 4 wherein the copolymer surfactant contains one or more hydrophobic reģions or domains selected from poly(meth)-acrylate esters, polyorthoesters, vinylic and styrenic polymers, polyacetals, polyanhydrides, polylactic acids, polyglycolic acids and ethers and esters thereof/ and polylactic acid/polyglycolic acid copolymers. 6. A contrast aģent as claimed in any of claims 2 to 4 wherein the copolymer surfactant contains one or more hydrophobic reģions or domains selected from polyester groups containing one or more long chain aliphatic groups. 38 7. λ contrast aģent as claimed in claim 6 wherein said long chain aliphatic group or groups are selected from polymethylene groups containing 10-20 carbon atoms. 8. A contrast aģent as claimed in any of the preceding claims wherein the copolymer surfactant is a block copolymer. 9. λ contrast aģent as claimed in claim 8 wherein the copolymer surfactant is an extended polymer. 10. A contrast aģent as claimed in claim 9 wherein said extended polymer comprises hydrophilic polymer blocks linked by oligomeric or quasi-polymeric hydrophobic reģions or domains. 11. λ contrast aģent as claimed in any of the preceding claims wherein the copolymer surfactant is biodegrad-able. 12. A contrast aģent as claimed in claim 11 wherein the copolymer surfactant contains acid-labile bonds. 13. A contrast aģent as claimed in claim 11 vherein the copolymer surfactant contains biodegradable linkages of formula - (Ojja-CO-O-CUlV) -O-CO- (0)n (I) 1 2 (where R and R each represent a hydrogen atom or a 1 2 carbon-attached monovalent organic group or R and R together form a carbon-attached divalent organic group, and m and n are each zero or 1). 14. A contrast aģent as claimed in claim 13 wherein R1 2 and R (when other than hydrogen) are selected from aliphatic groups having up to 10 carbon atoms, cycloalkyl groups having up to 10 carbon atoms, araliphatic groups having up to 20 carbon atoms, aryl groups having up to 20 carbon atoms, heterocyclic groups having up to 20 carbon atoms and one or more heteroatoms selected from Ο, N and S, and any of the preceding groups carrying one or more functional substituents. 39 LV 11438

15. A contrast aģent as claimed in claim 14 vherein R 2 and R are selected from hydrogen atoms and methyl groups. 16. A contrast aģent as claimed in any of claims 13 to 14 wherein the copolymer surfactant comprises one or more hydrophobic reģions or domains containing units of formula (I) . 17. A contrast aģent as claimed in any of claims 13 to 14 wherein the copolymer surfactant comprises one or more hydrophilic reģions or domains containing units of formula (I). 18. A contrast aģent.as claimed in any of the preceding claims further comprising at least one additional emulsifier. 19. A contrast aģent as claimed in claim 18 containing at least one emulsifier selected from fatty acids and carbohydrate and triglyceride esters thereof, phospho-lipids, proteins, polyethylene glycols and block copo-lymers. 20. A contrast aģent as claimed in any of the preceding claims which is coated with one or more coating materiāls selected from polyethylene glycols, proteins and polysaccharides. 21. A contrast aģent as claimed in any of the preceding claims wherein the encapsulated gas is selected from air, nitrogen, oxygen, hydrogen, nitrous oxide, carbon dioxide, helium, argon, sulphur hexafluoride and low molecular weight optionally fluorinated hydrocarbons. 22. A contrast aģent as claimed in claim 21 wherein the encapsulated gas is perfluorobutane or perfluoropentane. 23. Use of a contrast aģent as defined in any of claims 1 to 22 in diagnostic imaging. 24. Use of a contrast aģent as defined in any of claims 1 to 22 in diagnostic ultrasonic imaging. 25. Use of a contrast aģent as defined in any of claims 1 to 22 in magnetic resonance imaging. 40 26. A method ο£ generating enhanced images of a human or non-human animal body which comprises ādministering to said body a contrast aģent as defined in any of claims 1 to 22 and generating an ultrasound or MR image of at least a part of said body. 27. A process for the preparation of a contrast aģent as claimed in claim 1 which comprises interacting a gas with a non-polymerisable wall-£orming block or graft copolymer surfactant so as to producē said contrast aģent. 28. A process as claimed in claim 27 which comprises (i) generating an emulsion comprising hydrophilic and hydrophobic phases wherein the copolymer surfactant is preferentially solubilised in the dispersed phase thereof or is distributed about the interfaces between said phases and (ii) obtaining said contrast aģent from said emulsion. 29. A process as claimed in claim 28 wherein a multiple emulsion is prepared, the copolymer surfactant being preferentially solubilised in or distributed about the interfaces of the finest dispersed phase. 30. A process as claimed in claim 28 or 29 carried out in the presence of at least one additional emulsifier 31. A process as claimed in claim 30 carried but in the presence of at least one emulsifier selected from fatty acids and carbohydrate and triglyceride esters thereof, phospholipids, proteins, polyethylene glycols and block copolymers. 32. A process as claimed it claim 31 carried out in the presence of a poly (oxyethylene)-poly (oxypropylene) -poly(oxyethylene) block copolymer. 33. A process at claimed in any of claims 28 to 32 wherein the emulsion is lyophilised in the presence of the gas to be encapsulated. 34. A process as claimed in any of claims 27 to 33 wherein the contrast aģent is coated with one or more coating materiāls selected from polyethylene glycols, proteins and polysaccharides. 41 LV 11438 35. A process as claimed in claim 28 wherein the dispersed phase is gaseous and the copolymer surfactant is preferentially soluble in the dispersion phase, thereby generating a contrast aģent comprising a dispersion of gas microbubbles stabilised by encapsu- lating copolymer surfactant. 36. A process as claimed in claim 35 wherein the dispersed phase is a perfluoroalkane and the dispersion phase is an aqueous solution of a water-soluble copolymer surfactant. 37. A block or graft copolymer surfactant containing biodegradable linkages of formula - (O^-CO-O-COlV) -0-C0- (0)n (I) 1 2 (where R and R each represent a hydrogen atom or a 1 2 carbon-attached monovalent organic group or R and R together form a carbon-attached divalent organic group, and m and n are each zero or 1). 38. A copolymer surfactant as claimed in claim 37 1 2 wherein R and R (when other than hydrogen) are selected from aliphatic groups having up to 10 carbon atoms, cycloalkyl groups having up to 10 carbon atoms, araliphatic groups having up to 20 carbon atoms, aryl groups having up to 20 carbon atoms, heterocyclic groups to 20 carbon atoms and one or more heteroatoms selected from Ο, N and S, and any of the preceding groups carrying one or more functional substituents. 39. A copolymer surfactant as claimed in claim 38 1 2 wherein R and R are selected from hydrogen atoms and methyl groups. 40. A copolymer surfactant as claimed in claim 39 1 2 wherein R represents a hydrogen atom and R represents a methyl group. 41. A copolymer surfactant as claimed in any of claims 37 to 40 containing one or more hydrophilic reģions or domains and one or more hydrophobic reģions or domains. 42. A copolymer surfactant as claimed in claim 41 containing one or more hydrophilic reģions or domains 42 selected from polysaccharides, polyalcohols, polyvinyl-pyrrolidones, polethylene glycole and polyaminoacids. 43. Λ copolymer surfactant as claimed in claim 42 containing one or more hydrophilic reģions or domains consisting, essentially of polyethylene glycol units. 44. λ copolymer surfactant as claimed in any of claims 41 to 43 containing one or more hydrophobic reģions or domains selected from poly (meth.) acrylate esters, poly-orthoesters, vinylic and styrenic polymers, polyacetals, polyanhydrides, polylactic acidb, polyglycolic acids and ethers and esters thereof, and polylactic acid/poly-glycolic acid copolymers. 45. A copolymer surfactant as claimed in any of claims 41 to 43 containing one or more hydrophobic reģions or domains selected from polyester groups containing one or more long Chain aliphatic groups · 46. A copolymer surfactant as claimed in claim 45 wherein said long chain aliphatic group or groups are selected from polymethylene groups containing 10-20 carbon atoms. 47. λ copolymer surfacatant as claimed in any of claims 37 to 46 which is a block copolyjner. 48. A copolymer surtactant as claimed in claim 47 which is an extended polymer. 49. A copolymer surfactant a-s claimed in claim 48 comprising hydrophilic poly»*er blocks linked by oligomeric or guasi-polymeric hydrophobic domains or reģions. 50. A copolymer surfactant as claimed in any of claims 41 to 49 vherein one or more hydrophobic reģions or domains contain units of formula (I) as defined in claim 37. 51. A copolymer surfactant as claimed in any of claims 41 to 44 wherein one or more hydrophilic reģions or domains are water-insoluble and contain units of formula (I) as defined in claim 37. 43 LV 11438 52. A copolymer surfactant as claimed in any o£ claims 37 to 51 containing units of formula - (CH2) a- (OJ^CO-O-CIrV) -0-C0- (0)n- (CH2)b- (ii) (where R1, R2, m and n are as defined in claim 37 and a and b are each integers in the range 1-30). 53. A copolymer surfactant as claimed in claim 52 wherein a and b in formula (II) are each integers in the range 10-18. 54. An emulsion comprising a copolymer surfactant as claimed in any of claims 37 to 53 comprising hydrophilic and hydrophobic phases wherein the copolymer surfactant is preferentially solubilised in the dispersed phase thereof or is distributed about the interfaces between said phages. 55. Use of a copolymer surfactant as claimed in any of claims 37 to 53 or an emulsion thereof as claimed in claim 54 in drug formulations, drug delivery systems, contrast aģents, surgical materiāls, biocompatible implant surfaces, particle coatings, foods, paints, coatings, impregnations, cosmetics, detergents, textiles, coating of medical devices, biodegradable clear packaging film, polymer property modifiers, selflubricating materiāls, compatibilisers for polymer blends, separation membranes, anti-fouling coatings, foam forming materiāls, antifoams, phase transfer catalysts, thermoplastic elastomers or the manufacture of hydrogels. 56. PE6 2300 methyl ether 16-hexadecanoyloxyhexadecano-ate. 57. PEG 5000 methyl ether 16-hexadecanoyloxyhexadecano-ate. 58. PEG 10000 methyl ether 16-hexadecanoyloxyhexadeca-noate. 44

Claims (58)

1. A contrast agent comprised of encapsulated gas bladders, wherein the capsule walls are formed from non-polymerizable prepolymerized or block copolymer surfactant copolymers. 2. A contrast agent according to claim 1, wherein the surfactant copolymer comprises one or more hydrophilic domains or domains and one or more hydrophobic regions or domains. 3. A contrast agent according to claim 2, wherein the surfactant copolymer comprises one or more hydrophilic domains or domains taken from the group consisting of polysaccharides, polyhydric alcohols, polyvinylpyrrolidones, polyethylene glycols and peptides. 4. A contrast agent according to claim 3, wherein the surfactant copolymer comprises one or more hydrophilic domains or domains consisting exclusively of polyethylene glycol. 5. The contrast agent according to any one of claims 2 to 4, wherein the surfactant copolymer comprises one or more hydrophobic regions or domains taken from the group of: poly (meth) acrylic acid esters, polyester esters, polyvinyl compounds, polystyrene, polyacetal, polyanhydrides, lactic acid polymers, polyglycolic acids and their ethers and esters, and copolymers of lactic and glycolic acids. 6. A contrast agent as claimed in any one of claims 2 to 4, wherein the surfactant copolymer comprises one or more hydrophobic regions or domains made of polyesters containing one or more long aliphatic strands. 7. A contrast agent according to claim 6, wherein the long aliphatic chains are formed from polymethylene groups containing 10-20 carbon atoms. 1 8. A contrast agent according to any one of the preceding claims, wherein the surface-active copolymer is a block copolymer. 9. The contrast agent of claim 8, wherein the surfactant copolymer is a polymer diluted with a filler. 10. A contrast agent according to claim 9, wherein the filler-diluted polymer comprises hydrophilic polymer blocks joined by oligomers or quasi-polymers of hydrophobic regions or domains. 11. A contrast agent according to any one of the preceding claims, wherein the surfactant copolymer is biodegradable. 12. A contrast agent according to claim 11, wherein the surfactant copolymer comprises bonds which cleave under the action of an acid. 13. The contrast agent of claim 11, wherein the surfactant copolymer comprises biodegradable steps of formula (I) - (O) m-C0-0-C (R1 R2) -O-C0- (O) n- (I) (wherein R 1 and R 2 are independently hydrogen or a monovalent organic group attached to the carbon atom, or R 1 and R 2 together form a bivalent organic group attached to the carbon atom; m and n are independently of one another is zero or 1). 14. A contrast agent according to claim 13, wherein R1 and R2 (when not hydrogen) are taken from the group consisting of aliphatic groups containing up to 10 carbon atoms; cycloalkyl containing up to 10 carbon atoms; arylaliphatic groups containing up to 20 carbon atoms; aryl groups containing up to 20 carbon atoms; heterocyclic groups containing up to 20 carbon atoms and one or more heterocycles from row Ο, N and S; any of said groups with one or more functional substituents. 15. The contrast agent of claim 14, wherein R1 and R2 are hydrogen or methyl. 16. A contrast agent according to claim 13 or 14, wherein the surfactant copolymer comprises one or more hydrophobic regions or domains containing the steps of formula (I). 17. The contrast agent according to claim 13 or 14, wherein the surfactant copolymer comprises one or more hydrophilic domains or domains containing the steps of formula (I). 18. The composition of any one of the preceding claims, further comprising at least one emulsifier. 2 EN 11438 19. A contrast agent as claimed in claim 18, comprising at least one emulsifier from the fatty acids, their carbohydrate esters and triglycerides, phospholipids, obaltum, polyethylene glycols and block copolymers. 20. A congener according to any one of the preceding claims, characterized in that it is coated with one or more of a series of polyethylene glycols, proteins and polysaccharides. 21. A congener according to any one of the preceding claims, wherein the encapsulated gas is one of the series: air, nitrogen, oxygen, hydrogen, nitrogen oxide, carbon dioxide, helium, argon, sulfur hexafluoride and optionally fluorinated low molecular weight hydrocarbons. 22. The contrast agent of claim 21, wherein the encapsulated gas is perfluorobutane or perfluoropentane. Use of a contrast agent according to any one of the preceding claims for image acquisition in diagnostics. Use of a contrast agent according to any one of claims 1 to 22 for obtaining an image in ultrasound diagnostics. Use of a contrast agent according to any one of claims 1 to 22 for obtaining a picture in nuclear magnetic resonance diagnostics. 26. A method for obtaining a detailed image of a human or other animal body, characterized in that a contrast agent is administered to the body according to any one of claims 1 to 22 and obtains an image of at least a part of the body by ultrasound or magnetic resonance imaging. A method for obtaining a contrast agent according to claim 1, characterized in that the gas is contacted with a non-polymerizable prepolymer or block copolymer surfactant which forms the walls of said contrast medium capsules. 28. The method of claim 27, wherein: (i) an emulsion comprising a hydrophilic and a hydrophobic phase wherein the surfactant copolymer is substantially dissolved in the dispersed phase or concentrated on the phase boundary; (ii) emits a contrast agent from the emulsion. 29. A method according to claim 28, wherein a multicomponent emulsion is formed in which the surfactant copolymer is mainly dissolved in the most dispersed phase or concentrated on its surface. 30. The method of claim 28 or 29, wherein said method is carried out in the presence of at least one additional emulator. 31. A method according to claim 30, characterized in that it is carried out in the presence of at least one additional emulsifier selected from the group consisting of: 3 fatty acids, their carbohydrate esters and triglycerides, phospholipids, protein substances, polyethylene glycols and block copolymers. 32. The method of claim 31, wherein the process is carried out in the presence of a block copolymer of poly (oxyethylene) poly (oxypropylene) -poly (oxyethylene). A method according to any one of claims 28 to 32, wherein the emulsion is lyophilized in the presence of encapsulated gas. 34. A method according to any one of claims 27 to 33, wherein the contrast agent is coated with one or more of a series of polyethylene glycols, proteins and polysaccharides. 35. The method of claim 28, wherein the dispersed phase is a gaseous and surfactant copolymer is substantially soluble in the dispersion phase, thereby providing a contrast agent in which the microbubbles of the dispersed gas are stabilized by the surface-active copolymer forming the capsule. 36. The method of claim 35, wherein the dispersed phase is perfluoroalkane and the dispersion phase is a water-soluble surface-active copolymer aqueous solution. 37. A surfactant-coated or block copolymer, characterized in that it comprises biodegradable steps of formula (I) - (O) m -CO-OC (R1 R2) -O-C0- (O) - (I) ( wherein R 1 and R 2 are independently hydrogen or a monovalent organic group attached to the carbon atom, or R 1 and R 2 together form a bivalent organic group attached to the carbon atom, m and n independently of one another are zero or 1 ). 38. A surfactant copolymer according to claim 37, wherein R1 and R2 (when not hydrogen) are taken from the group consisting of: an aliphatic group containing up to 10 carbon atoms; cycloalkyl containing up to 10 carbon atoms; arylaliphatic groups containing up to 20 carbon atoms; aryl groups containing up to 20 carbon atoms; heterocyclic groups containing up to 20 carbon atoms and one or more heterocycles from row Ο, N and S; any of said groups with one or more functional substituents. 39. The surfactant copolymer of claim 38, wherein R1 and R2 are hydrogen or methyl. 40. A surfactant copolymer according to claim 39, wherein R1 is hydrogen and R2 is methyl. 41. A surfactant copolymer according to any one of claims 37 to 40, characterized in that it comprises one or more hydrophilic domains or domains and one or more hydrophobic regions or domains. 4 EN 11438 42. A surfactant copolymer according to claim 41, characterized in that it contains one or more hydrophilic regions or domains taken from the group of polysaccharides, polyhydric alcohols, polyvinylpyrrolidones, polyethylene glycols and peptides. 43. A surfactant copolymer according to claim 42, characterized in that it contains one or more hydrophilic regions or domains consisting solely of polyethylene glycol. 44. A surfactant copolymer as claimed in any one of claims 41 to 43, comprising one or more hydrophobic regions or domains taken from the group of: poly (meth) acrylic acid esters, polyester esters, polyvinyl compounds, polystyrene, polyacetal, polyanhydrides, lactic acid polymers, polyglycolic acids and their ethers and esters, and copolymers of lactic and glycolic acids. 45. A surfactant copolymer according to any one of claims 41 to 43, characterized in that it comprises one or more hydrophobic regions or domains formed from polyesters containing one or more long aliphatic strands. 46. The surfactant copolymer of claim 45, wherein the long aliphatic strands are composed of polymethylene groups containing from 10 to 20 carbon atoms. 47. A surfactant copolymer as claimed in any one of claims 37 to 46, characterized in that it is a block copolymer. 48. The surfactant copolymer of claim 47, characterized in that it is a polymer diluted with a filler. A surface active copolymer according to claim 48, characterized in that it comprises hydrophilic polymer blocks joined by oligomers or quasi-polymers of hydrophobic regions or domains. 50. A surfactant copolymer as claimed in any one of claims 41 to 49, wherein one or more hydrophobic regions or domains comprise steps having a structure according to formula (I) of claim 37. \ t 51. The surfactant copolymer of any one of claims 41 to 44, wherein one or more hydrophilic regions or domains are water-insoluble and comprises the steps of a structure corresponding to the formula (I) of claim 37. 52. The surfactant copolymer of any one of claims 37 to 51, comprising the steps of a structure corresponding to formula (II) - (CH2) a - (O) m-C0-0-C (R1R2). ) -O-C0- (O) n- (CH2) b- (II) (wherein R1, R2, m and n are as defined in item 37, and a and b are from 1 to 30). 5 53. The surfactant copolymer according to claim 52, wherein in formula (II) a and b are from 10 to 18. 54. An emulsion formed by a surfactant copolymer as claimed in any one of claims 37 to 53, comprising hydrophilic and hydrophobic phases, wherein the surfactant copolymer is substantially dissolved in the dispersed phase or concentrated on the phase boundary. 55. The use of a surface active copolymer according to any one of claims 37 to 53 or an emulsion thereof according to claim 54 in finished dosage forms, drug delivery systems, contrast media, dressings, biocompatible implanted surfaces, particle coatings, foodstuffs, paints, surface coatings, impregnation compositions, cosmetics, detergents, textiles, medical device coatings, biodegradable transparent packaging films, polymeric properties of modifying additives, self-lubricating materials, homogenisation additives for polymer blends, separation membranes, coatings to protect products from spoilage, foam forming materials, anti-foaming compositions , interphase catalysts, thermoplastic elastomers or hydrogel production. 56. Polyethylene glycol PEG 2300 methyl ether 16-hexadecanoyloxyhexane-decanoyl ester. 57. Polyethylene glycol PEG 5000 methyl ether 16-hexadecanoyloxyhexane decanoyl ester. 58. Polyethylene glycol PEG 10,000 methyl ether 16-hexadecanoyloxyhexane-decanoyl ester. 6